Kamran Harati

Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines

BackgroundTaurolidine (TRD) represents an anti-infective substance with anti-neoplastic activity in many malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by TRD simultaneously in different malignant cell lines.Materials and methodsFive different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma, AsPC-1/pancreas and BxPC-3/pancreas) were incubated with increasing concentrations of TRD (100 μM, 250 μM and 1000 μM) for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis (Propidiumiodide/AnnexinV staining). Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the radical scavenger N-Acetylcystein (NAC) and the Gluthation depleting agent BSO to examine the contribution of caspase activation and reactive oxygen species in TRD induced cell death.ResultsAll cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and BSO co-treatment were highly different among cell lines - suggesting a cell line specific involvement of ROS in TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly among the cell lines.ConclusionThis is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.

Upper extremity ischemia is superior to lower extremity ischemia for remote ischemic conditioning of antero-lateral thigh cutaneous blood flow.

Remote ischemic conditioning (RIC) is known to improve microcirculation in various settings, but little is known about the impact of the amount of ischemic tissue mass or the limb itself. Since ischemia and subsequent necrosis of flaps is one of the most dreaded complications in reconstructive surgery, adjuvant methods to improve microcirculation are desirable. We therefore performed a randomized trial to compare the effect of arm versus leg ischemia for RIC of the cutaneous microcirculation of the antero–lateral thigh. Forty healthy volunteers were randomized to undergo 5 min of ischemia of either the upper or lower extremity, followed by 10 min of reperfusion.Ischemia was induced by a surgical tourniquet applied to the proximal limb, which was inflated to 250 mmHg for the upper and 300 mgHg for the lower extremity. This cycle was repeated a total of three times. Cutaneous microcirculation was assessed by combined laser doppler spectrophotometry on the antero–lateral aspect of the thigh to measure cutaneous blood flow (BF), relative hemoglobin content (rHb), and oxygen saturation (StO2). Baseline measurements were performed for 10 min, after which the ischemia/reperfusion cycles were begun. Measurements were performed continuously and were afterwards pooled to obtain a mean value per minute. Both groups showed significant increases in all three measured parameters of cutaneous microcirculation after three cycles of ischemia/reperfusion when compared to baseline (BF: 95.1% (P < 0.001) and 27.9% (P = 0.002); rHb: 9.4% (P < 0.001) and 5.9% (P < 0.001), StO2: 8.4% (P = 0.045) and 9.4% (P < 0.001). When comparing both groups, BF was significantly higher in the arm group (P = 0.019 after 11 min., P = 0.009 after 45 min). In conclusions, both ischemic conditioning of the upper and lower extremity is able to improve cutaneous BF on the ALT donor site. However, RIC of the upper extremity seems to be a superior trigger for improvement of cutaneous BF.

Thoracic Wall Reconstruction after Tumor Resection

Introduction Surgical treatment of malignant thoracic wall tumors represents a formidable challenge. In particular, locally advanced tumors that have already infiltrated critical anatomic structures are associated with a high surgical morbidity and can result in full-thickness defects of the thoracic wall. Plastic surgery can reduce this surgical morbidity by reconstructing the thoracic wall through various tissue transfer techniques. Sufficient soft-tissue reconstruction of the thoracic wall improves quality of life and mitigates functional impairment after extensive resection. The aim of this article is to illustrate the various plastic surgery treatment options in the multimodal therapy of patients with malignant thoracic wall tumors. Materials and methods This article is based on a review of the current literature and the evaluation of a patient database. Results Several plastic surgical treatment options can be implemented in the curative and palliative therapy of patients with malignant solid tumors of the chest wall. Large soft-tissue defects after tumor resection can be covered by local, pedicled, or free flaps. In cases of large full-thickness defects, flaps can be combined with polypropylene mesh to improve chest wall stability and to maintain pulmonary function. The success of modern medicine has resulted in an increasing number of patients with prolonged survival suffering from locally advanced tumors that can be painful, malodorous, or prone to bleeding. Resection of these tumors followed by thoracic wall reconstruction with viable tissue can substantially enhance the quality of life of these patients. Discussion In curative treatment regimens, chest wall reconstruction enables complete resection of locally advanced tumors and subsequent adjuvant radiotherapy. In palliative disease treatment, plastic surgical techniques of thoracic wall reconstruction provide palliation of tumor-associated morbidity and can therefore improve patients’ quality of life.

Remote Ischemic Conditioning improves blood flow and oxygen saturation in pedicled and free surgical flaps.

Background: Surgical flaps have become safe and reliable reconstructive tools, but total flap loss rates as high as 25 percent and partial flap loss rates as high as 36 percent have been reported due to insufficient perfusion. Therefore, a reliable, noninvasive, and effective way to improve the microcirculation of surgical flaps is desirable. The aim of this study was to assess the effect of remote ischemic conditioning on the microcirculation of pedicled and free surgical flaps. Methods: Thirty patients undergoing free (n = 20) and pedicled (n = 10) tissue transfer were included in this study. Remote ischemic conditioning was applied on the upper extremity for three cycles on postoperative days 1, 5, and 12. Blood flow, tissue oxygen saturation, and relative hemoglobin content were measured by means of a combination of laser Doppler and spectroscopy (O2C device) in the flap and the surrounding tissue. The relative increase compared with baseline measurements was assessed. Results: Blood flow increased significantly in controls on all 3 postoperative days (p < 0.05 for all). In free flaps, tissue oxygen saturation improved significantly on postoperative days 1 and 12 and blood flow improved significantly on postoperative days 5 and 12 (p < 0.05). In pedicled flaps, blood flow and tissue oxygen saturation increased on postoperative day12, but not significantly. Conclusions: Remote ischemic conditioning is a safe, inexpensive, fast, and reliable method to improve the microcirculation of surgical flaps. Further research is warranted to see whether such an improvement translates into improved flap survival, but it is likely. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Effects of TRAIL and taurolidine on apoptosis and proliferation in human rhabdomyosarcoma, leiomyosarcoma and epithelioid cell sarcoma

Soft tissue sarcomas (STS) are a heterogeneous group of malignant tumours representing 1% of all malignancies in adults. Therapy for STS should be individualised and multimodal, but complete surgical resection with clear margins remains the mainstay of therapy. Disseminated soft tissue sarcoma still represents a therapeutic dilemma. Commonly used chemotherapeutic agents such as doxorubicin and ifosfamide have proven to be effective in fewer than 30% in these cases. Therefore, we tested the apoptotic and anti-proliferative in vitro effects of TNF-related apoptosis-inducing ligand (TRAIL) and taurolidine (TRD) on rhabdomyosarcoma (A-204), leiomyosarcoma (SK-LMS-1) and epithelioid cell sarcoma (VA-ES-BJ) cell lines. Viability, apoptosis and necrosis were quantified by FACS analysis (propidium iodide/Annexin V staining). Gene expression was analysed by DNA microarrays and the results validated for selected genes by rtPCR. Protein level changes were documented by western blot analysis. Cell proliferation was analysed by BrdU ELISA assay. The single substances TRAIL and TRD significantly induced apoptotic cell death and decreased proliferation in rhabdomyosarcoma and epithelioid cell sarcoma cells. The combined use of TRAIL and TRD resulted in a synergistic apoptotic effect in all three cell lines, especially in rhabdomyosarcoma cells leaving 18% viable cells after 48 h of incubation (p<0.05). Analysis of the differentially regulated genes revealed that TRD and TRAIL influence apoptotic pathways, including the TNF-receptor associated and the mitochondrial pathway. Microarray analysis revealed remarkable expression changes in a variety of genes, which are involved in different apoptotic pathways and cross talk to other pathways at multiple levels. This in vitro study demonstrates that TRAIL and TRD synergise in inducing apoptosis and inhibiting proliferation in different human STS cell lines. Effects on gene expression differ relevantly in the sarcoma entities. These results provide experimental support for in vivo trials assessing the effect of TRAIL and TRD in STS and sustain the approach of individualized therapy.

Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

BackgroundThe anti-infective agent Taurolidine (TRD) has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types.MethodsFive different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3) were incubated with TRD (100 μM, 250 μM and 1000 μM). Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot.ResultsTRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3) as well as genes involved in the ER stress response (PPP1R15A), in ubiquitination (TRAF6) and mitochondrial apoptotic pathways (PMAIP1).ConclusionsThis is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis.

Surgical Debridement Is Superior to Sole Antibiotic Therapy in a Novel Murine Posttraumatic Osteomyelitis Model.

Introduction Bone infections after trauma, i.e. posttraumatic osteomyelitis, pose one of the biggest problems of orthopedic surgery. Even after sufficient clinical therapy including vast debridement of infected bone and antibiotic treatment, regeneration of postinfectious bone seems to be restricted. One explanation includes the large sized defects resulting from sufficient debridement. Furthermore, it remains unclear if inflammatory processes after bone infection do affect bone regeneration. For continuing studies in this field, an animal model is needed where bone regeneration after sufficient treatment can be studied in detail. Methods For this purpose we created a stable infection in murine tibiae by Staphylococcus aureus inoculation. Thereafter, osteomyelitic bones were debrided thoroughly and animals were subsequently treated with antibiotics. Controls included debrided, non-infected, as well as infected animals exclusively treated with antibiotics. To verify sufficient treatment of infected bone, different assessments detecting S. aureus were utilized: agar plates, histology and RT-qPCR. Results All three detection methods revealed massive reduction or eradication of S. aureus within debrided bones 1 and 2 weeks postoperatively, whereas sole antibiotic therapy could not provide sufficient treatment of osteomyelitic bones. Debrided, previously infected bones showed significantly decreased bone formation, compared to debrided, non-infected controls. Discussion Thus, the animal model presented herein provides a reliable and fascinating tool to study posttraumatic osteomyelitis for clinical therapies.

Reconstruction of Soft-Tissue Defects at the Foot and Ankle after Oncological Resection

Introduction Solid malignancies at the foot and ankle region are rare and include mainly soft-tissue sarcomas, bone sarcomas, and skin malignancies. Complete surgical resection with clear margins still remains the mainstay of therapy in these malignancies. However, attainment of negative surgical margins in patients with locally advanced tumors of the foot and ankle region may require extensive surgery and could result in loss of extremity function. In these circumstances, plastic surgical techniques can frequently reduce functional impairment and cover soft-tissue defects, particularly in cases of large tumor size or localization adjacent to critical anatomic structures, thereby improving the quality of life for these patients. The aim of this article is to illustrate the various treatment options of plastic surgery in the multimodal therapy of patients with malignant tumors of the foot and ankle region. Materials and methods This article is based on the review of the current literature and the evaluation of the author’s own patient database. Results The local treatment of malignant extremity tumors has undergone major changes over the last few decades. Primary amputations have been increasingly replaced by limb-sparing techniques, preserving extremity function as much as possible. Although defect coverage at the foot and ankle region is demanding due to complex anatomical features and functional requirements, several plastic surgical treatment options can be implemented in the curative treatment of patients with malignant solid tumors in this area. Soft-tissue defects after tumor resection can be covered by a variety of local flaps. If local flaps are not applicable, free flap transfers, such as the anterolateral thigh flap, parascapular flap, or latissimus dorsi flap, can be utilized to cover nearly all kinds of defects in the foot and ankle region. Conclusion Soft-tissue reconstruction in the foot and ankle region is a vital component of limb-sparing surgery. It enables complete resection of locally advanced tumors and subsequent adjuvant radiotherapy. Modern plastic surgical techniques should, therefore, be integrated in the multimodal treatment concept of malignancies in the foot and ankle region.

Anti‑proliferative activity of epigallocatechin‑3‑gallate and silibinin on soft tissue sarcoma cells

Disseminated soft tissue sarcomas (STS) present a therapeutic dilemma. The first-line cytostatic doxorubicin demonstrates a response rate of 30% and is not suitable for elderly patients with underlying cardiac disease, due to its cardiotoxicity. Well-tolerated alternative treatment options, particularly in palliative situations, are rare. Therefore, the present study assessed the anti-proliferative effects of the natural compounds epigallocatechin-3-gallate (EGCG), silibinin and noscapine on STS cells. A total of eight different human STS cell lines were used in the study: Fibrosarcoma (HT1080), liposarcoma (SW872, T778 and MLS-402), synovial sarcoma (SW982, SYO1 and 1273) and pleomorphic sarcoma (U2197). Cell proliferation and viability were analysed by 5-bromo-2′-deoxyuridine and MTT assays and real-time cell analysis (RTCA). RTCA indicated that noscapine did not exhibit any inhibitory effects. By contrast, EGCG decreased proliferation and viability of all cell lines except for the 1273 synovial sarcoma cell line. Silibinin exhibited anti-proliferative effects on all synovial sarcoma, liposarcoma and fibrosarcoma cell lines. Liposarcoma cell lines responded particularly well to EGCG while synovial sarcoma cell lines were more sensitive to silibinin. In conclusion, the green tea polyphenol EGCG and the natural flavonoid silibinin from milk thistle suppressed the proliferation and viability of liposarcoma, synovial sarcoma and fibrosarcoma cells. These compounds are therefore potential candidates as mild therapeutic options for patients that are not suitable for doxorubicin-based chemotherapy and require palliative treatment. The findings from the present study provide evidence to support in vivo trials assessing the effect of these natural compounds on solid sarcomas.

Local Application of Isogenic Adipose-Derived Stem Cells Restores Bone Healing Capacity in a Type 2 Diabetes Model

Bone regeneration is typically a reliable process without scar formation. The endocrine disease type 2 diabetes prolongs and impairs this healing process. In a previous work, we showed that angiogenesis and osteogenesis—essential steps of bone regeneration—are deteriorated, accompanied by reduced proliferation in type 2 diabetic bone regeneration. The aim of the study was to improve these mechanisms by local application of adipose‐derived stem cells (ASCs) and facilitate bone regeneration in impaired diabetic bone regeneration. The availability of ASCs in great numbers and the relative ease of harvest offers unique advantages over other mesenchymal stem cell entities. A previously described unicortical tibial defect model was utilized in diabetic mice (Leprdb−/−). Isogenic mouse adipose‐derived stem cells (mASCs)db−/db− were harvested, transfected with a green fluorescent protein vector, and isografted into tibial defects (150,000 living cells per defect). Alternatively, control groups were treated with Dulbeccos modified Eagles medium or mASCsWT. In addition, wild‐type mice were identically treated. By means of immunohistochemistry, proteins specific for angiogenesis, cell proliferation, cell differentiation, and bone formation were analyzed at early (3 days) and late (7 days) stages of bone regeneration. Additionally, histomorphometry was performed to examine bone formation rate and remodeling. Histomorphometry revealed significantly increased bone formation in mASCdb−/db−‐treated diabetic mice as compared with the respective control groups. Furthermore, locally applied mASCsdb−/db− significantly enhanced neovascularization and osteogenic differentiation. Moreover, bone remodeling was upregulated in stem cell treatment groups. Local application of mACSs can restore impaired diabetic bone regeneration and may represent a therapeutic option for the future.